Specifically, we propose a Competitive Revision Application for our active grant 2R01CA098727 "Cell Biology of Retrovirus Replication". The ability of retroviruses to efficiently manipulate cell-cell contacts for the purpose of spreading critically contributes to the progression to retrovirally induced diseases such as leukemia and AIDS. Our grant 2R01CA098727 is devoted to the determination of the mechanism by which retroviruses establish and manipulate cell-cell contact for the purpose of efficient spreading from cell to cell. Here we propose a Competitive Revision Application to establish the experimentally conditions needed to directly visualize, for the first time, retroviral spreading in vivo using intravital two-photon microscopy. The proposed approach is within reach due to two important technical breakthroughs. First, we have identified an HIV variant as the first fluorescently labeled retrovirus that can be seen by two-photon microscopy. Second, new humanized mouse models provide a small animal model for HIV infection that is accessible to intravital imaging. Both advances allow us to develop the technologies that permit the visualization of HIV spreading in vivo using the humanized mouse model of HIV infection. Our grant will overcome the limitation associated with in vitro studies and has the potential to revolutionize our understanding of retroviral spreading as it happens in living organism. PUBLIC HEALTH RELEVANCE: This Competitive Revision Application for the active grant 2R01CA098727 is in response to NOT-OD-09-058 and proposes the establishment of the experimentally conditions needed to directly visualize, for the first time, retroviral spreading in vivo using intravital two-photon microscopy. The proposal will overcome the limitation associated with in vitro studies and will significantly elevate the quality, scope and relevance of the active grant.